Acute and Sub-Chronic Toxicity Studies of Aqueous Extract of Root Bark of Cassia Sieberiana D . C . in Rodents

Article history: Received on: 28/02/2014 Revised on: 02/04/2014 Accepted on: 22/04/2014 Available online: 28/04/2014 Acute and sub-chronic toxicity studies of the aqueous extract of the root bark of C. sieberiana (Caesalpiniaceae), a plant used locally in Ghana for the treatment of pain, was carried out in rodents. In the acute study, a single oral dose (5000 mg/kg) of the aqueous extract of C. sieberiana (NPK) was administered to six rats and six mice, and observed for 14 days for signs of acute toxicity. In the sub-chronic study, rats were administered with NPK (15750 mg/kg) daily for three months. Urinalysis, haematological and biochemical analyses were carried out on urine, blood and serum samples collected at the end of the three-month treatment. Histological analyses of the liver, heart, kidney and lung tissues were also done. The results showed that administration of 5000 mg/kg of NPK to animals did not result in death. There were no significant differences (p>0.05) between control and test animals in the haematological assay. The albumin, alkaline phosphatase and total bilirubin were higher (p<0.05) in test animals compared to the controls. Liver micrographs showed centrilobular necrosis at the dose of 750 mg/kg. The findings, therefore, show that the oral toxicity of NPK in rodents is low (oral LD50 > 5000 mg/kg). However, the extract may have deleterious effects on the liver at high doses on prolonged administration.


INTRODUCTION
Cassia sieberiana DC. (Caesalpiniaceae), commonly called African laburnum is a perennial savannah legume shrub or tree, up to 20 m high.The leaves are spirally arranged, leaflets elliptical to ovate, shortly hairy.The fruit is a cylindrical pod (up to 90 cm) which contains ellipsoid, rusty to dark brown seeds (van der Maesen, 2007).The aqueous decoctions of the roots, stem bark and the fruit pulp have been used traditionally in North-eastern Nigeria for the treatment of inflammatory conditions, tiredness, jaundice and joint pains (Madusolomuo et al., 1999).The extracts are used to treat fever, malaria, diuretics, diarrhoea, leprosy, bilhazia, stomach pains and as a dewormer (Tamboura et al., 2005).Presently, at the Centre for scientific Research into Plant Medicine (CSRPM), Mampong-Akwapim, Ghana, the milled root bark of the plant is processed and used for the treatment of various pain conditions including pain associated with stomach ulcer and menstruation.Earlier works have shown that C. sieberiana extracts had antimicrobial, antifungal, anti-oxidant and anti-ulcerogenic activity (Silva et al., 1997a;Silva et al., 1997b;Asase et al., 2008;Nartey et al., 2012).
Ethanolic and aqueous root extracts of C. sieberiana also has analgesic, anti-inflammatory, antiparasitc, myorelaxant and antispasmodic activities (Duwiejua et al., 2008;Sy et al., 2009).The anti-nociceptive activity of the ethyl acetate extract of the root bark has been shown to be attributable to interactions with opioidergic, muscarinic cholinergic and adenosinergic systems (Donkor et al., 2013).
The phytochemical studies of the plant indicated the presence of tannins, flavonoids, sterols, saponins, phenolics and anthroquinones (Duquenois et al., 1968;Madusolomuo et al., 1999;Donkor et al., 2013).Obidah and his co-workers (Obidah et al., 2009) reported that oral administration of aqueous extract of C. sieberiana stem bark to rats resulted in hepatotoxicity at lower dose levels of 20-60 mg/kg and nephrotoxicity at higher doses of 180 mg /kg.Earlier, Tamboura et al. (Tamboura et al., 2005) found that the LD 50 of the aqueous leaf extract in mice was 24.4 mg/kg.
There are other reports of toxicity of C. Sieberiana and other members of its genera in literature (Dhar et al., 1968;Mugera, 1970;El Sayed et al., 1983;Neuwinger, 1996).Though the root bark of the plant is widely used medicinally, little studies has been done on its toxicity.Only an acute single-dose toxicity study has been reported (Nartey et al., 2012)-which recorded no sign of toxicity up to a dose level of 2000 mg/kg body weight p.o.There is, therefore, the need for a thorough safety assessment of the root bark of the plant.In this regard, we have examined, in the current study, the acute and sub-chronic toxicity of the aqueous extract of the root bark of C. sieberiana in rodents.

Plant material
The root bark of Cassia sieberiana was collected from the CSRPM's arboretum (5°55'05.44"N;0°07´58.95"W) at Mampong-Akwapim, Ghana.They were authenticated by Mr. Ofori Lartey of the Plant Development Department (PDD) of the CSRPM and a voucher specimen (CSRPM No. 300) kept in the Herbarium of the PDD of the CSRPM, Mampong-Akwapim, Ghana.

Preparation of Extract
A kilogram of milled air-dried root bark of the plant material was boiled and kept at a temperature of 80 °C for 3 hours.It was left to cool at room temperature and sieved through a fine mesh.The mixture was lyophilized to yield 301.52 g (30.15% w/w of raw plant material) and labelled NPK.

Animals
Female Sprague-Dawley rats (190-205 g) and Female C3H mice (26-30 g) were obtained from the Animal Unit of the Centre for Scientific Research into Plant Medicine (CSRPM), Mampong-Akuapem, in the Eastern Region of Ghana.The animals were housed in groups of six in stainless steel cages (34×47×18 cm 3 ) with soft wood shavings as bedding, fed with normal commercial pellet diet (GAFCO, Tema), given clean water ad libitum and maintained under laboratory conditions (temperature 24-28 °C, relative humidity 60-70 %, and 12 hour light-dark cycle).The research was conducted in accordance with the internationally accepted principles for laboratory animal use and care (NIH, No.85-23, revised 1985).Ethical clearance was obtained from the CSRPM's ethics committee.

Acute toxicity studies
A single oral dose of the extracts was administered at 5.0 g /kg body weight to six female rats and six female mice each with an oral gavage needle.Mortality and general behaviour of the animals were observed over a 48-hour period.Surviving animals were observed for a further period of 12 days for toxic symptoms of piloerection, as well as lachrymatory, locomotor and respiratory activities.

Sub-chronic toxicity studies
Four groups of six female rats each were kept in four separate metal cages.Group 1 was kept as control and received normal clean drinking water ad libitum for three months.Group 2, 3 and 4 were treated with 15, 150 and 750 mg/kg of NPK daily for three months.The animals in each group were weighed on day zero (baseline) and weekly thereafter.Blood samples of rats in each treatment group was obtained by tail bleeding, at baseline and termination of treatment, into Eppendorf tubes without anticoagulant, centrifuged (Denley BS 400, England) at 4000 ×g for 5 min and the serum obtained stored at -40 °C for biochemical analyses.Other blood samples were collected into separate tubes already coated with trisodium citrate (Westergreen E.S.R, UK) for haematological analysis.

Urinalysis
Urine samples of rats in each treatment group at baseline and termination of treatment were analysed for glucose, bilirubin, ketones, specific gravity, pH, proteins, urobilinogen, nitrate, blood and leukocytes using urine reagent strips (UroColor TM 10, Standard Diagnostic Inc., Korea).

Histology
At termination of treatment, two rats from each group were sacrificed by cervical dislocation and the heart, lungs, liver, kidney and spleen were excised and weighed.All the organs except the spleen were stored in 10 % neutral formalin and dehydrated with a progressively increasing concentration of ethanol.The tissues were cleared with chloroform and impregnated with paraffin wax.Sections, 5 µm thick were stained with haematoxylin and eosin (Khalaf et al., 2010) and mounted on slides for light microscopic examinations.

Statistical analysis
Results were expressed as the mean value ± standard error of mean (SEM).Differences between control and experimental groups were determined by one-way analysis of variance (ANOVA), followed by Holm-Sidak's multiple comparisons test.GraphPad Prism for Windows version 6 (GraphPad Software, San Diego, CA, USA) was used for all statistical analyses and values less than 0.05 were considered significant.

Acute toxicity studies
The administration of 5000 mg/kg to both rats and mice did not result in the death of any of the animals for the 14-day experimental period.Also, animals did not show signs of acute toxicity such as piloerection, lacrimation or changes in locomotion and respiration.

Sub-chronic toxicity studies Body and organ weights
The percentage change in mean body weight with period of treatment, and organ/body weight ratio at termination of treatment in control and NPK-treated animals are shown in Fig. 1 and Table 1, respectively.The results showed no significant difference (p>0.05) in body weight changes between NPK-treated and control animals with time.Similarly, there were no significant changes (p>0.05) in the organ weights, expressed as percentage of body weight, at termination between control and NPK-treated animals..17 289 ± 42.79 497.33 ±107.72 645 ± 36.77 MPV (µ3 ) 6.55 ± 0.18 6.90 ± 0.12 7.00 ± 0.16 6.78 ± 0.14 LYMPH (#) 6.82 ± 0.77 6.32 ± 0.25 7.75 ± 0.69 8.15 ± 0.36 Values are means ± S.E.M. of N = 6.

Serum biochemistry and urinalysis
The effects of sub-chronic administration of NPK to rats on selected serum biochemical and urine parameters at termination of treatment are shown in Tables 2 and 3.The results of serum biochemical indices indicate that NPK caused significant increases (p<0.05) in the levels of ALB, ALP and total bilirubin compared to the controls.Dipstick urinalysis data indicate that there were no significant differences (p>0.05) in the levels of urine parameters between NPK-treated and control animals except ketones where there was a significant reduction (p<0.01) in the levels between test and control animals.

Haematological studies
The effect of sub-chronic administration of NPK on certain haematological indices at termination of treatment is shown in (Table 4).The results show that there were no significant difference (p>0.05) in all parameters measured between control and NPK-treated animals.

Histology
The effects of NPK on the histopathology of the liver, kidney, lung and heart tissues at termination of treatment are shown in Figs.3-6.Results showed that NPK did not affect the morphology of the kidney and heart tissues (Figs.4a-d, 6a-d).However, lungs of 15 and 150 mg/kg NPK-treated animals showed slight inflammatory response in alveolar areas and Clara cell hyperplasia without the thickening of alveolar septa and bronchiolar epithelial wall (Figs.5a-d).Similarly, there was centrilobular necrosis of the 750 mg/kg NPK-treated animals (Figs.3a-d)

DISCUSSION
In Ghana, the herbal medicine industry is becoming highly lucrative as a result of increased patronage of herbal preparations.Aside the government agency, Centre for Scientific Research into Plant Medicine, (CSRPM), Mampong-Akwapim, whose mandate is to carry out research and development of herbal medicine, there are numerous other private companies which are into the production and administration of herbal medicines.The CSRPM currently produces more than forty herbal preparations which it administers in its clinic including milled root bark of Cassia sieberiana (NPK).Despite the increased patronage of herbal preparations, information on their safety is very scanty.Thus we have evaluated comprehensively, the acute and subchronic toxicity of NPK in animals.
The limit dose of 5000 mg/kg did not result in mortality or any clinical sign of acute toxicity in both mice and rats in the short term (i.e., 48 h) and long term (i.e., 14 days) observatory period suggesting that the oral LD 50 of the extract is more than 5000 mg/kg in both species.The limit test is used in situations where the investigator has information indicating that the test material is likely to be non-toxic or of low toxicity (OECD, 2001).In a previous study, the oral LD 50 of root bark of C. sieberiana in rats was estimated to be more than 2000 mg/kg (Nartey et al., 2012).The result of the acute oral toxicity study, therefore, suggests that NPK at the limit dose tested is essentially non-toxic (scale of Hodge and Sterner(Hodge et al., 1943)), and safe in aqueous oral formulation in rodents.
In the sub-chronic studies, the extract did not appear to affect the normal growth of test animals as evidenced by comparable weight gain by both control and test animals over the three month treatment period (fig.1).Furthermore, there was no significant change when excised heart, kidney, liver, lung and spleen were expressed relative to body weight in control and test animals (Table .1).Relative weight is more indicative of toxicity than absolute weight (Demma et al., 2007).
Liver and kidney play a key role in metabolic processes.Whiles the liver detoxifies substances that are harmful to the body, the kidney helps in maintenance of homeostasis by reabsorbing vital substances and excretion of waste products (Greaves, 2007).The biochemical analysis (Table 2) showed a significant elevation in serum total bilirubin, ALB (at 750 mg/kg) and ALP (at 150 mg/kg) in NPK-treated animals compared to controls.In the assessment of liver damage by drugs or any other hepatotoxin, the determination of enzyme levels such as ALT and AST is largely used (Dobbs et al., 2003).Serum ALP level on the other hand, is related to the function of hepatic cell and increased level is due to increased synthesis of the enzyme, in the presence of increasing biliary pressure (Moss et al., 1974).Albumin is synthesised in the liver-decreased levels are implicated in malfunction in hepatic synthetic ability whiles excessive levels are usually due to dehydration (Pritchett, 2009).Thus the elevated levels of ALP and total bilirubin in NPK-treated animals may be indicative of liver dysfunction as a result of prolonged administration of the extract.Urea is the first acute renal marker upon renal injury and creatinine is the most trustable renal marker and increases only when the majority of renal function is lost (Borges et al., 2005).The present result reveals a reduction, though not statistically significant, in the levels of urea and creatinine at all the dose levels compared to controls.Nephrotoxicants such as cadmium increase serum levels of urea and creatinine in the rat whiles nephroprotective agents such as honey reduces serum urea and creatinine levels (Abdel-Moneim et al., 2007).Results of the urinalysis revealed no significant differences between test and control animals in all the parameters measured (Table 3).
Blood cells are produced in the bone marrow.Known haemotoxicants such as paracetamol cause reduction in RBCs leading to anaemia (Mullick et al., 1973) and some bioactive phytochemicals affect HCT levels (Patrick-Iwuanyanwu et al., 2007).In the haematological studies (Table 4), there was no significant difference between test and control animals.Hence, the extract may not have deleterious effects on bone marrow function.Histological examination of the extract-treated samples of liver stained with haematoxylene and eosin revealed slight centrilobular necrosis at the highest dose of 750 mg/kg.Known hepatotoxins such as paracetamol cause hepatocyte degeneration, centrilobular necrosis and haemorrhages (Iweala et al., 2011).Thus the extract may have a deleterious effect on the liver at high dose of 750 mg/kg on prolonged administration as evidenced by centrilobular necrosis and elevation in the serum ALP levels.The kidney and heart did not show noticeable morphological changes in all the NPK-treated animals.However, there was some toxicological insult to the lungs as evidenced by slight inflammatory response and Clara cell hyperplasia in the NPK-treated animals.
In conclusion, oral toxicity of the aqueous extract of the root bark of C. sieberiana in rodents is low (oral LD 50 > 5000 mg/kg).However, the extract may have deleterious effects on the liver at high doses on prolonged administration.These findings support its safe ethnomedicinal use at moderate doses.

Fig. 1 :
Fig. 1: Effect of oral administration of NPK on the body weight of rats in the sub-chronic toxicity test.Data are expressed as Mean ± S.E.M. (N=6).Treated groups were compared to controls using a two-way ANOVA (treatment × time) followed by Holm-Sidak's test.

Fig. 5 :
Fig. 5: Heart sections (H&E-stained 132×) of control rats (a) and rats treated daily with NPK at doses of 15 mg/kg (b), 150 mg/kg (c) and 750 mg/kg (d) for three months in the sub-chronic toxicity test.

Table . 1
: Effect of NPK on organ/body weight ratio.

Table . 2
: Effect of NPK on serum biochemical parameters.

Table . 4
: Effect of NPK on haematological parameters.